1. Technical Field
The present invention relates generally to oligonucleotide functionalizing reagents, and more particularly relates to novel reagents for the introduction of sulfhydryl, amino and hydroxyl groups into synthetic oligonucleotides.
2. Description of the Prior Art
Non-isotopically labeled synthetic DNA fragments have found broad application in molecular biology--e.g., in the areas of DNA sequencing, DNA probe-based diagnostics, and the like. The reagents disclosed herein facilitate the labeling of oligonucleotides with specific groups by incorporating one or more unique, modifiable sulfhydryl, amino or hydroxyl groups within the oligonucleotide at any position, typically at the 5' terminus.
Several references teach methods of introducing a sulfhydryl or an amino group at the 5' terminus of synthetic oligonucleotides. For example, Connolly, in Nuc. Acids Res. 13(12): 4485-4502 (1985) and in Nuc. Acids Res. 15(7): 3131-3139 (1987), describes a method of incorporating a sulfhydryl moiety into synthetic DNA using S-trityl-O-methoxy-morpholinophosphite derivatives of 2-mercaptoethanol, 3-mercaptopropan-1-ol and 6-mercaptohexan-1-ol--i.e., reagents given by the formula ##STR1## where x is 2, 3 or 6. Connolly further describes derivatization of the sulfhydryl-containing oligonucleotides with thiol-specific probes.
Coull et al., in Tetrahedron Lett. 27(34): 3991-3994 (1986), describe a reaction which incorporates an aliphatic primary amino group at the 5' terminus of oligonucleotides using an N-protected aliphatic amino phosphoramidite given by the structure ##STR2## as the functionalizing reagent; Sproat et al., Nuc. Acids Res. 15(15): 6181-6196 (1987), describes a similar method. Smith et al., in Nuc. Acids Res. 13(7): 2399-2411 (1985), also describes a method for synthesizing oligonucleotides containing a 5' aliphatic amino group, by direct reaction of oligonucleotides with protected phosphoramidite derivatives of 5'-amino-5'-deoxythymidine. An additional functionalizing reagent for introducing primary amines at the 5' terminus is that sold under the trademark "Aminolink" by Applied Biosystems, Inc., and given by the formula ##STR3## This reagent requires treatment with the activating agent dimethylaminopyridine prior to use and also necessitates deprotection with thiophenol, a sensitive, malodorous reagent.
These and other prior art methods suffer from one or more of the following disadvantages:
(1) A short spacer chain linking the 5' terminus of the oligonucleotide to the sulfhydryl, amino or hydroxyl group results in destabilization of the derivatived structure--i.e., proximity of a solid support or a bulky labeling species to the oligonucleotide chain causes steric interference and thus hinders use of the derivatived oligonucleotide in probe-based applications;
(2) A hydrophobic spacer chain linking the 5' terminus of the oligonucleotide to the sulfhydryl, amino or hydroxyl group provides problems with solubility in the aqueous solvents commonly used in DNA probe-based methods;
(3) Conventionally used functionalizing reagents are often incompatible with commonly used DNA synthesis methodology, primarily because the functionalizing reagents are incompatible with the reagents and solvents typically used therewith;
(4) Conventionally used functionalizing reagents are frequently difficult to synthesize in high yield, necessitating complex, multi-step reactions;
(5) As noted above in the case of Aminolink, certain known reagents require treatment with multiple activating agents immediately prior to use;
(6) Conventionally used functionalizing reagents do not allow for "tacking on" of multiple spacer chains to increase the distance between the terminal sulfhydryl, amino or hydroxyl moiety and the oligonucleotide chains, nor, generally, do they allow for multiple functionalization along an oligonucleotide chain;
(7) Conventionally used functionalizing reagents do not generally allow for functionalization at positions other than at the 5' hydroxyl terminus; and
(8) Conventionally used functionalizing reagents sometimes require deprotection under harsh conditions, in such a way that, frequently, the deprotection reaction is not readily monitorable.
There is thus a need in the art for oligonucleotide functionalizing reagents which address the aforementioned considerations.